Methods and compositions for improved cognition

ABSTRACT

Provided herein are klotho polypeptide compositions and methods for improving cognitive function in an individual comprising treatment of with klotho polypeptides.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 16/751,108, filed Jan. 23, 2020, which is a continuation ofU.S. patent application Ser. No. 15/548,306, filed Aug. 2, 2017, nowissued as U.S. Pat. No. 10,632,180 on Apr. 28, 2020, which is a U.S.National Phase Application of PCT/US2016/016842, filed Feb. 5, 2016,which claims benefit of priority to U.S. Provisional Patent ApplicationNo. 62/113,300, filed Feb. 6, 2015, each of which are incorporated byreference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with government support under grant no. K08AG034531 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

REFERENCE TO SUBMISSION OF A SEQUENCE LISTING AS A TEXT FILE

The Sequence Listing written in file 081906_1209930_Sequence_Listing.txtcreated on Nov. 9, 2020, 12,288 bytes, machine format IBM-PC, MS-Windowsoperating system, is hereby incorporated by reference in its entiretyfor all purposes.

BACKGROUND OF THE INVENTION

Brain health is one of the biggest biomedical challenges with few if anyeffective medical treatments. Cognition is a highly valued and centralmanifestation of brain health that is impaired or becomes disrupted innormal aging, numerous neurodegenerative, neurologic, and psychiatricdiseases, childhood developmental syndromes, traumatic brain injury, andstress. Cognition is also disrupted by jet lag, medication side effects,and certain medical treatments, such as those for cancer. Thus, thepotential to enhance cognition or counter cognitive dysfunction is ofenormous relevance across the human lifespan in health and disease.

BRIEF SUMMARY OF THE INVENTION

Provided herein are methods and compositions for improving cognitionthrough systemic administration of klotho or a protein comprising klothoor a functional fragment thereof. In some embodiments, the methodcomprises improving cognition in an individual comprising administeringto the individual an effective amount of a protein comprising a Klothopolypeptide or a functional variant or fragment thereof, therebyimproving cognitive function in the individual. In some embodiments, theadministering is systemic, peripheral, or nasal. In some embodiments,the protein is the Klotho polypeptide or a fragment thereof. In someembodiments, the administering is oral, mucosal, or carried out byinjection. In some embodiments, the injection is intravenous,intraperitoneal, subcutaneous, or intramuscular. In some embodiments,the administration is by infusion, e.g., continuous infusion using areservoir or osmotic minipump.

In some embodiments, the individual is a human. In some embodiments, thehuman has at least normal cognitive function and the administeringresults in improved cognitive function compared to before theadministering. In some embodiments, the human is 50 years of age orolder (e.g., at least 60, 65, 70, 75, 80, 85, 90, 100, or older). Insome embodiments, the individual is experiencing age-related cognitivedecline. In some embodiments, the administering results in a reducedrate of cognitive decline, e.g., so that short term memory does notdecline as quickly as expected based on pre-treatment or age-cohortdecline. In some embodiments, the individual is less than 50 years ofage.

In some embodiments, the individual has a neurodegenerative disease,e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease,frontotemporal dementia, progressive supranuclear palsy, corticobasalardegeneration, mild cognitive impairment, vascular dementia, Lewy bodydementia, multiple system atrophy, amyotropic lateral sclerosis, priondisorder, and HIV-related dementia. In some embodiments, the individualhas a mental or mood disorder, e.g., depression, schizophrenia,attention deficit/hyperactivity disorder, autism spectrum disorder,intellectual disability, a mood disorder, or a psychotic disorder. Insome embodiments, the individual has a condition selected from traumaticbrain injury, stroke, multiple sclerosis, neuroautoimmune disease,epilepsy, delirium, and a paraneoplastic disorder. In some embodiments,the individual has a condition selected from X-linked mental disorder,Down's syndrome, Angelman's syndrome, and Rett's syndrome. In someembodiments, the individual has a condition selected fromphenylketonuria, Lesch-Nyhan, galactosemia, and adrenoleukodystrophy. Insome embodiments, the individual is receiving radiation treatment orchemotherapy for cancer. In some embodiments, the individual isexperiencing or is expected to experience (e.g., in about 2 hours to 2weeks, about 12-48 hours, or about 24 hours) stress, pain, sleepdeprivation, or jet lag.

In some embodiments, the human has impaired motor function theadministering results in improved motor function compared to before theadministering.

In some embodiments, the klotho polypeptide has at least 75% identity(e.g., at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%identity) to soluble human klotho (amino acids 34-979 of SEQ ID NO:1).In some embodiments, the klotho polypeptide is a functional fragmentcomprising a polypeptide with at least 75% identity (e.g., at least 80,85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity) to the KL1domain of human klotho. In some embodiments, the klotho polypeptide is afunctional fragment comprising a polypeptide with at least 75% identity(e.g., at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%identity) to the KL2 domain of human klotho. In some embodiments, theklotho polypeptide retains at least 40, 50, 60, 70, 80, 90, or 100% ofthe level of at least one activity of soluble human klotho.

In some embodiments, the klotho polypeptide is administered to theindividual at a dose of 0.1-50,000 μg/kg body weight (e.g., 0.5-10μg/kg, 0.5-500 μg/kg, 1-2000 μg/kg, 1-250 μg/kg, 5-250 μg/kg, 10-100μg/kg, 1000-20,000 μg/kg, or about 10, 25, 50, 100, or 1000 μg/kg). Insome embodiments, the klotho polypeptide is administered daily, twiceper week, weekly, or every two weeks. In some embodiments, the klothopolypeptide is administered prior to (e.g., 2, 6, 12, 24, or 48 hours)or in response to an event requiring heightened cognition, e.g., stress,jet lag, sleep deprivation, or anticipated taxing mental task.

In some embodiments, the individual is tested for cognitive abilityprior to the administering. In some embodiments, the individual istested for cognitive ability after the administering. In someembodiments, the individual is tested for cognitive ability before andafter the administering, or multiple times during the course oftreatment. In some embodiments, the individual is tested for semantic,episodic, procedural, priming, and/or working memory. In someembodiments, the individual is tested for language ability, executivefunction, visuospatial function, or dementia. In some embodiments, thedose or frequency of administration of klotho polypeptide is increasedwhen cognitive ability does not significantly increase.

In some embodiments, a method of improving motor function is provided.In some embodiments, a method is provided for improving motor functionin an individual in need thereof, the method comprising administering tothe individual an effective amount of a protein comprising a Klothopolypeptide or a functional fragment thereof, wherein the administeringis systemic or peripheral, thereby improving motor function in theindividual compared to before the administering. In some embodiments,the klotho polypeptide has at least 75% identity (e.g., at least 80, 85,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity) to solublehuman klotho (amino acids 34-979 of SEQ ID NO:1). In some embodiments,the klotho polypeptide is a functional fragment comprising a polypeptidewith at least 75% identity (e.g., at least 80, 85, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, or 100% identity) to the KL1 domain of human klotho.In some embodiments, the klotho polypeptide is a functional fragmentcomprising a polypeptide with at least 75% identity (e.g., at least 80,85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity) to the KL2domain of human klotho. In some embodiments, the klotho polypeptideretains at least 40, 50, 60, 70, 80, 90, or 100% of the level of atleast one activity of soluble human klotho. In some embodiments, theklotho polypeptide is administered to the individual at a dose of0.1-50,000 μg/kg body weight (e.g., 0.5-10 μg/kg, 0.5-500 μg/kg, 1-2000μg/kg, 1-250 μg/kg, 5-250 μg/kg, 10-100 μg/kg, 1000-20,000 μg/kg, orabout 10, 25, 50, 100, or 1000 μg/kg). In some embodiments, the klothopolypeptide is administered daily, twice per week, weekly, or every twoweeks.

In some embodiments, the individual with impaired motor function hasstroke to the brain or spinal cord (ischemic or hemorrhagic),neurodegenerative disease (Parkinson's disease, Lewy body dementia,multiple system atrophy, amyotropic lateral sclerosis, prion disorder,Huntington's disease, supranuclear palsy), Parkinsonism, traumatic braininjury, neuroinfectious brain lesions, multiple sclerosis and relatedautoimmune and demyelinating disease, spinal cord lesions (compressive,infectious, toxic or metabolic, autoimmune, oncologic), brain tumor,epilepsy, paraneoplastic disorder, neurodevelopmental disorder(mitochondrial, autosomal genetic), muscle disease (polymyositis,dermatomyositis, inclusion body myositis, infectious, endocrine,metabolic, toxic, congenital myopathy, congenital muscular dystrophy,hereditary), neuropathies (Guillain-Barre syndrome, axonal anddemyelinating, diabetic, toxic, metabolic, infectious, critical illness,entrapment), tick paralysis, myasthenia gravis, and spinal muscularatrophy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-J. Klotho delivery enhances cognition in mice. Mice were testedin cognitive tasks following i.p. delivery of vehicle (Veh) orrecombinant mouse klotho (KL) (10 μg/kg) (age 4 months). Data aremean±SEM.

FIG. 1A. Mice were tested in the Y maze 18 hrs after treatment withvehicle or klotho. Percent alternations among arms during 4 minutes ofexploration of a Y maze are shown (n=4 male mice/group).

FIG. 1B-J. Mice were tested in the Morris water maze after dailytreatment with vehicle or klotho (two independent cohorts shown: Cohort1 and Cohort 2).

FIG. 1B. Spatial learning curves when the platform is hidden are shownin Cohort 1. Veh or KL was administered 4 h prior to testing. Datarepresent the daily average of total distance traveled on Days 1-4 toreach the hidden platform. Day 0 represents distance traveled on thefirst trial of Day 1. On Day 5, when the platform was visible, Veh- andklotho-treated mice located it equally well. (n=4 male mice/group).

FIG. 1C. Veh- and klotho-treated mice swam at equal speeds in Cohort 1,as measured by the average velocity from hidden training on Day 4.

FIG. 1D. Spatial learning curves when the platform is hidden are shownin an independent cohort of mice, Cohort 2. Veh or KL was administered18 h prior to testing. Data represent the daily average of distancetraveled on Days 1-4 to reach the hidden platform. (n=7-10 mice/group;males and females included)

FIG. 1E-J. Results of probe trials to assess spatial memory when theplatform was removed 1 hr and 24 hrs after completion of hidden-platformtraining in Veh- and klotho-treated mice in Cohort 1.

FIG. 1E. Duration at the target center in a 1 hr probe shows time spentat the original platform location.

FIG. 1F. Target platform crossings in a 1 hr probe shows the frequencyof crossings over the original platform location.

FIG. 1G. Percent time spent in the target quadrant, compared to theaverage time spent in other quadrants, in a 1 hr probe. *p<0.05 (t-test)

FIG. 1H. Duration at the target center in a 24 hr probe.

FIG. 1I. Target platform crossings in a 24 hr probe.

FIG. 1J. Percent time spent in the target quadrant in a 24 hr probe.

FIG. 2A-B. Acute delivery of klotho enhances cognition in aged mice.Mice were tested for spatial and working memory in the large Y-Mazefollowing i.p. delivery of vehicle (Veh) or recombinant mouse klotho(KL) (10 μg/kg) 24 hours prior to training (n=8-9 mice per experimentalgroup, sex-balanced groups, age 18 months, from NIH colony of agingmice). 18 hours after training, mice underwent testing and duration oftime spent in the novel arm and the familiar arm was measured duringexploration of the maze. (FIG. 2A) Novel arm preference is shown as aratio time spent in the novel compared to the familiar arm throughoutindicated times of exploration and (FIG. 2B) at 3 minutes. *p<0.05(t-test). Data are mean±SEM.

FIG. 3 . Acute delivery of klotho induces long-lasting cognitiveenhancement. Mice were tested for spatial and working memory in thelarge Y-Maze at 16 days following the last i.p. delivery of vehicle(Veh) or recombinant mouse klotho (KL) (0.5 or 2.5 μg/kg) that was givendaily for 5 days (n=8-10 mice per experimental group, sex-balancedgroups, age 5-5.5 months of age). Duration of time spent in the novelarm and the familiar arm was measured during exploration of the maze 18hours after training. Novel arm preference is shown as a ratio of timespent in the novel compared to the familiar arm at 5 min of exploration.**p=0.01 (Bonferroni-Holm test). Data are mean±SEM.

FIG. 4A-B. Acute delivery of klotho improves cognitive deficits intransgenic hSYN mice that express the human α-synuclein protein. Micewere tested for spatial and working memory in the large Y-Maze followingi.p. delivery of vehicle (Veh) or recombinant mouse klotho (KL) (2.5μg/kg) at 22 hours prior to training and then 14 hours prior to testing(n=6-9 male mice per group, age 2.5-6 months of age). Duration of timeand number of entries in the novel and familiar arms were measuredduring exploration of the maze 18 hours after training. Data aremean±SEM.

FIG. 4A. Novel arm preference is shown as ratio of time spent in thenovel compared to the familiar arm at 5 min of exploration. Two-wayANOVA: hSYN effect p=0.026, KL effect p=0.07; *p<0.05, #p=0.07(Bonferroni-Holm test)

FIG. 4B. Novel arm preference is shown as ratio of entries into thenovel compared to the familiar arm at 5 min of exploration. Two-wayANOVA: hSYN effect p=0.07, KL effect p=0.03; #p=0.07 (t-test).

FIG. 5 . Acute delivery of klotho improves early motor deficits intransgenic hSYN mice that express the human α-synuclein protein. Micewere tested for motor function on the rotarod task following i.p.delivery of vehicle (Veh) or recombinant mouse klotho (KL) (2.5 μg/kg)at approximately 17 hours prior to testing (n=6-10 male mice perexperimental group, age 2.5-6 months of age). Time spent on the spinningrod without falling is depicted in seconds (s). In hSYN mice,KL-treatment improves early motor function as shown by increasedduration of the average time spent on the spinning rod during Trials 1through 3. Two-way ANOVA: hSYN effect 0.0025; **p<0.01, *p<0.05(Bonferroni-Holm test). Data are mean±SEM.

FIG. 6 . Acute delivery of klotho does not alter the overall activity ofmice. Mice were tested for level of overall movements and total activityin the open field task at 16 hours following i.p. delivery of vehicle(Veh) or recombinant mouse klotho (KL) (2.5 μg/kg) (n=8 mice perexperimental group; sex-balanced groups; age 5 months). Total movementsover 5 minutes are depicted. Data are mean±SEM.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

Klotho is a relatively large protein, approximately 130 kD in itssecreted form, and is not expected to cross the blood-brain barrier. Theresults provided herein, however, show that Klotho exerts a positiveeffect on cognition within hours of systemic administration and longafter its half-life elimination. These results are highly unexpected,and provide the advantage of safer, more convenient therapies comparedto administration directly to the brain or into the cerebrospinal fluid.

II. Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by a person of ordinaryskill in the art. See, e.g., Lackie, DICTIONARY OF CELL AND MOLECULARBIOLOGY, Elsevier (4th ed. 2007); Sambrook et al., MOLECULAR CLONING, ALABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, N Y1989). Any methods, devices and materials similar or equivalent to thosedescribed herein can be used in the practice of this invention. Thefollowing definitions are provided to facilitate understanding ofcertain terms used frequently herein and are not meant to limit thescope of the present disclosure.

The terms “klotho” or “klotho polypeptide” refer to soluble klothopolypeptide, and functional variants and fragments thereof, unlessotherwise stated. Soluble klotho is any form of klotho that circulatesin fluid (e.g., serum, cerebrospinal fluid, etc.), and that does notinclude a transmembrane or intracellular component. Klotho can becleaved from its transmembrane form and released into fluid, orotherwise secreted or shed from a cell. Klotho RNA can also bealternatively spliced and directly secreted into the surrounding fluid(i.e., without forming a transmembrane protein). Both forms areencompassed in the terms soluble klotho polypeptide, klotho polypeptide,and klotho.

As used herein, the terms “systemic” or “peripheral” refer toadministration by a route that does not involve direct injection (orother administration) into the cerebrospinal fluid (CSF) or centralnervous system (CNS). That is, systemic and peripheral administrationencompasses administration to the “blood” side of the blood-brainbarrier. Examples of systemic and peripheral routes include oral andmucosal, intravenous, intraperitoneal, intramuscular, and subcutaneousinjection, and intravenous drip.

The terms “cognition,” “cognitive ability,” “cognitive function,” andlike terms refer to a collection of mental tasks and functions,including but not limited to: memory (e.g., semantic, episodic,procedural, priming, or working); orientation; language; problemsolving; visual perception, construction, and integration; planning;organizational skills; selective attention; inhibitory control; andability to mentally manipulate information.

The terms “improved cognition,” “increased cognitive ability,” “improvedcognitive function,” and like terms refer to an improvement in cognitionunder a given condition (e.g. treatment with klotho) compared tocognition absent the condition (e.g., absent treatment with klotho). Foran individual experiencing cognitive decline, an improvement incognition might be a reduction in the rate of cognitive decline (i.e.,an improvement compared to the absence of treatment), but not an actualimprovement in cognitive ability. An increase in cognitive ability canalso be an increase in brain activity in a specified area, e.g., asdetermined by MRI, or an inhibition of brain activity that results inbetter overall brain function. An increase in cognitive ability can alsobe improvement in a cognitive performance test as described in moredetail herein. An improvement or increase in cognitive ability can be inany one cognitive aspect or function, or any combination of individualcognitive functions.

An individual in need of improved cognitive function refers toindividuals with age-related cognitive decline; a neurodegenerativedisease; a mental or mood disorder; traumatic brain injury;developmental delay; genetic disorder resulting in reduced cognitiveability; brain injury due to stroke, brain cancer, MS, epilepsy,radiation or chemotherapy; etc. An individual in need of improvedcognitive function can also include individuals that desire increasedmental function to fight the effects of stress, sleep deprivation, jetlag, or pain, or to heighten ability for a particular task. A morecomplete and specific list of such individuals in included in the“Cognitive conditions and disorders” section herein.

The words “protein”, “peptide”, and “polypeptide” are usedinterchangeably to denote an amino acid polymer or a set of two or moreinteracting or bound amino acid polymers. The terms apply to amino acidpolymers in which one or more amino acid residue is an artificialchemical mimetic of a corresponding naturally occurring amino acid, aswell as to naturally occurring amino acid polymers, those containingmodified residues, and non-naturally occurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction similarly to the naturally occurring amino acids. Naturallyoccurring amino acids are those encoded by the genetic code, as well asthose amino acids that are later modified, e.g., hydroxyproline,γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers tocompounds that have the same basic chemical structure as a naturallyoccurring amino acid, e.g., an a carbon that is bound to a hydrogen, acarboxyl group, an amino group, and an R group, e.g., homoserine,norleucine, methionine sulfoxide, methionine methyl sulfonium. Suchanalogs may have modified R groups (e.g., norleucine) or modifiedpeptide backbones, but retain the same basic chemical structure as anaturally occurring amino acid. Amino acid mimetics refers to chemicalcompounds that have a structure that is different from the generalchemical structure of an amino acid, but that functions similarly to anaturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical or associated, e.g., naturallycontiguous, sequences. Because of the degeneracy of the genetic code, alarge number of functionally identical nucleic acids encode mostproteins. For instance, the codons GCA, GCC, GCG and GCU all encode theamino acid alanine. Thus, at every position where an alanine isspecified by a codon, the codon can be altered to another of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent variations,” which are onespecies of conservatively modified variations. Every nucleic acidsequence herein which encodes a polypeptide also describes silentvariations of the nucleic acid. One of skill will recognize that incertain contexts each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, often silent variations of a nucleicacid which encodes a polypeptide is implicit in a described sequencewith respect to the expression product, but not with respect to actualprobe sequences.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention. The following amino acids aretypically conservative substitutions for one another: 1) Alanine (A),Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine(L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (see, e.g., Creighton, Proteins (1984)).

The terms “identical” or “percent identity,” in the context of two ormore nucleic acids, or two or more polypeptides, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of nucleotides, or amino acids, that are the same (i.e.,about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specifiedregion, when compared and aligned for maximum correspondence over acomparison window or designated region) as measured using a BLAST orBLAST 2.0 sequence comparison algorithms with default parameters, or bymanual alignment and visual inspection. See e.g., the NCBI web site atncbi.nlm.nih.gov/BLAST. Such sequences are then said to be“substantially identical.” This definition also refers to, or may beapplied to, the compliment of a nucleotide test sequence. The definitionalso includes sequences that have deletions and/or additions, as well asthose that have substitutions. As described below, the algorithms canaccount for gaps and the like. Typically, identity exists over a regioncomprising an antibody epitope, or a sequence that is at least about 25amino acids or nucleotides in length, or over a region that is 50-100amino acids or nucleotides in length, or over the entire length of thereference sequence.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all.

The term “heterologous” when used with reference to portions of aprotein or nucleic acid indicates that the protein or nucleic acidcomprises two or more subsequences that are not found in the samerelationship to each other in nature. For instance, the protein ornucleic acid is typically recombinantly produced, having two or moresequences from unrelated genes arranged to make a new functional nucleicacid, e.g., a promoter from one source and a coding region from anothersource, or functional chimeric protein.

The terms “agonist,” “activator,” “inducer” and like terms refer to anagent that increases activity or expression (e.g., of klotho or a klothosignaling pathway) as compared to a control. Agonists are agents that,e.g., stimulate, increase, activate, enhance activation, sensitize orupregulate the activity of klotho or a klotho signaling pathway. Theexpression or activity can be increased 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90% 100% or more than that in a control. In certain instances,the activation is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, ormore in comparison to a control.

The terms “inhibitor,” “repressor” or “antagonist” or “downregulator”interchangeably refer to a substance that results in a detectably lowerexpression or activity level as compared to a control. The inhibitedexpression or activity can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90% or less than that in a control. In certain instances, the inhibitionis 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more incomparison to a control.

A “control” sample or value refers to a sample that serves as areference, usually a known reference, for comparison to a test sample.For example, a test sample can be taken from a test condition, e.g., inthe presence of a test compound, and compared to samples from knownconditions, e.g., in the absence of the test compound (negativecontrol), or in the presence of a known compound (positive control). Acontrol can also represent an average value gathered from a number oftests or results. One of skill in the art will recognize that controlscan be designed for assessment of any number of parameters. For example,a control can be devised to compare therapeutic benefit based onpharmacological data (e.g., half-life) or therapeutic measures (e.g.,comparison of benefit and/or side effects). Controls can be designed forin vitro applications. One of skill in the art will understand whichcontrols are valuable in a given situation and be able to analyze databased on comparisons to control values. Controls are also valuable fordetermining the significance of data. For example, if values for a givenparameter are widely variant in controls, variation in test samples willnot be considered as significant.

A “label” or a “detectable moiety” is a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, chemical, orother physical means. For example, useful labels include ³²P,fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonlyused in an ELISA), biotin, digoxigenin, or haptens and proteins or otherentities which can be made detectable, e.g., by incorporating aradiolabel into a peptide or antibody specifically reactive with atarget peptide. Any method known for conjugating a protein to the labelmay be employed, e.g., using methods described in Hermanson,Bioconjugate Techniques 1996, Academic Press, Inc., San Diego.

A “labeled” molecule (e.g., klotho polypeptide) is one that is bound,either covalently, through a linker or a chemical bond, ornoncovalently, through ionic, van der Waals, electrostatic, or hydrogenbonds to a label such that the presence of the molecule may be detectedby detecting the presence of the label bound to the molecule.

The term “diagnosis” refers to a relative probability that a disorder ispresent in an individual. Similarly, the term “prognosis” refers to arelative probability that a certain future outcome may occur in theindividual. For example, in the context of the present disclosure,prognosis can refer to the likelihood that an individual suffercognitive decline, or the likely severity of the disease (e.g., severityof symptoms, rate of functional decline, etc.). The terms are notintended to be absolute, as will be appreciated by any one of skill inthe field of medical diagnostics.

A “biological sample” can be obtained from a patient, e.g., a biopsy,from an animal, such as an animal model, or from cultured cells, e.g., acell line or cells removed from a patient and grown in culture forobservation. Biological samples include tissues and bodily fluids, e.g.,cerebrospinal fluid (CSF), blood, blood fractions, lymph, saliva, urine,feces, etc.

The terms “therapy,” “treatment,” and “amelioration” refer to anyreduction in the severity of symptoms (cognitive decline), orimprovement in cognitive function, or where motor function is affected,an improvement in motor function. As used herein, the terms “treat” and“prevent” are not intended to be absolute terms. Treatment andprevention can refer to any delay in cognitive decline, amelioration ofsymptoms (e.g., confusion, delirium), etc. Treatment and prevention canbe complete or partial, such that cognition is better than would beexpected without treatment (e.g., compared to cognition in the sameindividual before treatment or compared to cognition in similarnon-treated individuals). The effect of treatment can be compared to anindividual or pool of individuals not receiving the treatment, or to thesame patient prior to treatment or at a different time during treatment.In some aspects, cognition is improved by at least 1%, as compared,e.g., to the individual before administration or to a control individualnot undergoing treatment. In some embodiments, cognition is improved byat least 2, 3, 5, 7, 10, 15, 20, 25%, 50%, 75%, 80%, or 90%, or more,determined using tests of cognition, molecular proxies, or structuralchanges associated with brain function. In some aspects, motor functionis improved by at least 1%, as compared, e.g., to the individual beforeadministration or to a control individual not undergoing treatment. Insome embodiments, motor function is improved by at least 2, 3, 5, 7, 10,15, 20, 25%, 50%, 75%, 80%, or 90%, or more, determined using tests ofmotor function.

The terms “effective amount,” “effective dose,” “therapeuticallyeffective amount,” etc. refer to that amount of the therapeutic agentsufficient to ameliorate a disorder, as described above. For example,for the given parameter, a therapeutically effective amount will show anincrease or decrease of therapeutic effect at least 1%, 2%, 5%, 10%,15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%.Therapeutic efficacy can also be expressed as “-fold” increase ordecrease. For example, a therapeutically effective amount can have atleast a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over acontrol.

As used herein, the term “pharmaceutically acceptable” is usedsynonymously with physiologically acceptable and pharmacologicallyacceptable. A pharmaceutical composition will generally comprise agentsfor buffering and preservation in storage, and can include buffers andcarriers for appropriate delivery, depending on the route ofadministration.

The terms “dose” and “dosage” are used interchangeably herein. A doserefers to the amount of active ingredient given to an individual at eachadministration. For the present invention, the dose refers to the amountof Klotho polypeptide. The dose will vary depending on a number offactors, including frequency of administration; size and tolerance ofthe individual; type and severity of the condition; risk of sideeffects; and the route of administration. One of skill in the art willrecognize that the dose can be modified depending on the above factorsor based on therapeutic progress. The term “dosage form” refers to theparticular format of the pharmaceutical, and depends on the route ofadministration. For example, a dosage form can be in a liquid, e.g., asaline solution for injection.

“Subject,” “patient,” “individual” and like terms are usedinterchangeably and refer to, except where indicated, mammals such ashumans and non-human primates, as well as dogs, horses, pigs, mice,rats, and other mammalian species. The term does not necessarilyindicate that the subject has been diagnosed with a particular disease,but typically refers to an individual under medical supervision. Apatient can be an individual that is seeking treatment, monitoring,adjustment or modification of an existing therapeutic regimen, etc.

III. Klotho

Klotho is a pleiotropic protein and an aging regulator that circulatesthroughout the body and brain (Imura et al. (2004) FEBS Letters 565:143;Kurosu et al. (2005) Science 309:1829). Human Klotho is described inGenBank Accession No. NC_000013 and Uniprot Accession No. Q9UEF7. Anumber of species homologs exist, including mouse and rat Klotho whichshare 86% and 85% identity with the human Klotho polypeptide, which isshown as SEQ ID NO:1. It exists in a transmembrane form that can becleaved such that the extracellular portion (amino acids 34-979) isreleased as a hormone (Shiraki-lika et al. (1998) FEBS Letters 424:6).Klotho also has a splice variant that results in a 549 amino acidsecreted form of the protein that is also functional (Wang and Sun(2009) Ageing Res. Rev. 8:43). Both cleaved and secreted klotho aresoluble and functional in the body, but have a sequence variation at theC-terminal end due to the splice variation. Amino acids 535-549 areDTTLSQFTDLNVYLW (SEQ ID NO:2) for cleaved, soluble human Klotho andSQLTKPISSLTKPYH (SEQ ID NO:3) for spliced, soluble human Klotho. Fulllength soluble Klotho includes two conserved domains (KL1 and KL2) withhomology to beta glycosidase proteins. The conservedbeta-glucosidase/6-phospho-beta-glucosidase/beta-galactosidase motifspans 62-497 in the human protein and 64-499 in the mouse. The conservedKL1 sequence is described in Chateau et al. (2010) Aging 2:567 andMatsumura et al. (1998) Biochem Biophys Res Commun, and comprises aminoacids 34-549 of the human Klotho protein, with the glycosyl hydrolaseconsensus region spanning amino acids 57-506 of the human Klotho protein(59-508 of the mouse). Klotho does not have beta-glycosidase activity,but shows some beta-glucuronidase activity.

Klotho suppresses insulin and wnt signaling, regulates ion channels andtheir transport, and promotes function of FGF23. See, e.g., Chang et al.(2005) Science 310:490; Imura et al. (2007) Science 316:1615; Kurosu(2005); Liu et al. (2007) Science 317:803; and Urakawa et al. (2006)Nature 444:770).

In mice, transgenic overexpression of klotho extends lifespan andassociates with better cognitive functions in the normal and diseasedbrain (Kurosu (2005); Dubal et al. (2014) Cell Reports 7:1065; Dubal etal. (2015) J Neuroscience). In humans, a single allele of the KL-VSvariant of the KLOTHO gene, which increases secreted klotho promoteslongevity (Arking et al., 2002; Arking et al., 2005; Invidia et al.,2010) and also associates with better baseline cognitive functions inaging populations. See, e.g., Arking et al. (2002) PNAS 99:856; Arkinget al. (2005) Circ. Res. 96:412; Dubal et al (2014) Cell Reports 7:1065;Yokoyama et al. (2015) Ann. Clin. Translational Neurology 2:215.

Klotho polypeptides that can be used for administration include specieshomologs (e.g., non-human primate, mouse, rat), allelic variants,functional fragments, and functional variants of the wild type sequencethat retain cognition improving activity. Examples include secretedKlotho, fragments comprising the KL1 domain, fragments comprising theKL2 domain, fragments comprising the KL1 and KL2 domains, variantscomprising the KL1 domain with at least one (e.g., 1-20, 5-50, 25-100)non-conserved amino acid in the KL1 domain substituted with a differentamino acid or deleted, variants comprising the KL2 domain with at leastone non-conserved amino acid in the KL2 domain substituted with adifferent amino acid.

Functional fragments of the Klotho polypeptide that can be used asdescribed herein include the extracellular domain (e.g., correspondingto or substantially identical or similar to amino acids 34-979 of humanKlotho), secreted Klotho (e.g., corresponding to or substantiallyidentical or similar to 549 amino acid form), a KL1 domain (e.g.,corresponding to or substantially identical or similar to amino acids34-549 of human Klotho), a glycosyl hydrolase consensus sequence (e.g.,corresponding to or substantially identical or similar to amino acids57-506 of human Klotho), or abeta-glucosidase/6-phospho-beta-glucosidase/beta-galactosidase consensussequence (e.g., corresponding to or substantially identical or similarto amino acids 62-497 of human Klotho). In some embodiments, the Klothopolypeptide comprises or is substantially identical or similar to aminoacids 34-549 of human Klotho. In some embodiments, the Klothopolypeptide is part of a larger fusion protein. In some embodiments, thefusion protein comprises the Klotho polypeptide as described herein andfurther comprises no more than 100, 75, 50, or 30 additional aminoacids. In some embodiments, the Klotho polypeptide is not fused to aFibroblast growth factor (FGF). In some embodiments, the Klothopolypeptide comprises (e.g., is fused to) an affinity tag (e.g., ahistidine tag) or a conjugate to increase stability or half-life invivo.

A functional variant or fragment of Klotho is a variant or fragment thatretains any klotho activity, e.g., at least 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, or 100% of the level of any activity of soluble klotho.Soluble klotho activities include those described above, and includebinding FGF-23, binding to FGFR1c, beta-glucuronidase activity,suppression of wnt signaling, suppression of insulin signaling,suppression of TFG-beta 1 activity, increasing GluN2B expression and/orsynaptic localization, c-fos induction. Additional Klotho activitiesinclude causing changes in magnetic resonance imaging (MRI) brain scans,e.g., functional MRI, electroencephalograph (EEG), and transcranialmagnetic and electrical stimulation (TMS and TES); and improvedperformance in neuropsychologic testing and cognitive ability.

IV. Administration of Klotho

Provided herein are methods of improving cognition and/or motor functionin an individual comprising administering Klotho to the individual. Insome embodiments, the method of treatment comprises administering to anindividual an effective amount of a Klotho polypeptide (or functionalvariant or fragment thereof). In some embodiments, the treatment isprophylactic, e.g., for individual expecting stress (e.g., jet lag,military performance) or to prevent cognitive decline associated withaging. In some embodiments, the individual has been diagnosed with acognitive disorder. In some embodiments, the individual is receiving orhas received therapy for a cognitive disorder or for a condition that isrelated to cognitive function (e.g., cognitive decline in response tochemotherapy).

In some embodiments, the method further comprises monitoring theindividual for cognitive ability, either through a molecular proxy(e.g., changes NMDA receptor or c-fos activation, or GluN2B levels inthe brain), changes in MRI brain scans (e.g., functional MRI), changesin EEG, changes in TMS and TES, changes in neuropsychologic test scores,or tests of cognitive ability (e.g., for learning, short or long termmemory, executive functions, language ability, and visuospatialfunction). In some embodiments, the individual is monitored using morethan one of the above tests in any combination. In some embodiments, thedose of the Klotho polypeptide for each administration is determinedbased on the therapeutic progress of the individual, e.g., where ahigher dose is administered if the individual is not respondingsufficiently to therapy.

In some embodiments, the Klotho polypeptide is administered in apharmaceutical composition with a physiologically (i.e.,pharmaceutically) acceptable carrier. The term “carrier” refers to atypically inert substance used as a diluent or vehicle for a diagnosticor therapeutic agent. The term also encompasses a typically inertsubstance that imparts cohesive qualities to the composition.Physiologically acceptable carriers can be liquid, e.g., physiologicalsaline, phosphate buffer, normal buffered saline (135-150 mM NaCl),water, buffered water, 0.4% saline, 0.3% glycine, glycoproteins toprovide enhanced stability (e.g., albumin, lipoprotein, globulin, etc.),and the like. Since physiologically acceptable carriers are determinedin part by the particular composition being administered as well as bythe particular method used to administer the composition, there are awide variety of suitable formulations of pharmaceutical compositions ofthe present invention (See, e.g., Remington's Pharmaceutical Sciences,17^(th) ed., 1989).

The presently described compositions can be sterilized by conventional,well-known sterilization techniques or may be produced under sterileconditions. Aqueous solutions can be packaged for use or filtered underaseptic conditions and lyophilized, the lyophilized preparation beingcombined with a sterile aqueous solution prior to administration. Thecompositions can contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions, such aspH adjusting and buffering agents, tonicity adjusting agents, wettingagents, and the like, e.g., sodium acetate, sodium lactate, sodiumchloride, potassium chloride, calcium chloride, sorbitan monolaurate,and triethanolamine oleate. Sugars can also be included for stabilizingthe compositions, such as a stabilizer for lyophilized antibodycompositions.

Dosage forms can be prepared for mucosal (e.g., nasal, sublingual,vaginal, buccal, or rectal), parenteral (e.g., subcutaneous,intravenous, intramuscular, or intraarterial injection, either bolus orinfusion), oral, or transdermal administration to a patient. Examples ofdosage forms include, but are not limited to: dispersions;suppositories; ointments; cataplasms (poultices); pastes; powders;dressings; creams; plasters; solutions; patches; aerosols (e.g., nasalsprays or inhalers); gels; liquid dosage forms suitable for oral ormucosal administration to a patient, including suspensions (e.g.,aqueous or non-aqueous liquid suspensions, oil-in-water emulsions, or awater-in-oil liquid emulsions), solutions, and elixirs; liquid dosageforms suitable for parenteral administration to a patient; and sterilesolids (e.g., crystalline or amorphous solids) that can be reconstitutedto provide liquid dosage forms suitable for parenteral administration toa patient.

Injectable compositions can comprise a solution of the Klothopolypeptide suspended in an acceptable carrier, such as an aqueouscarrier. Any of a variety of aqueous carriers can be used, e.g., water,buffered water, 0.4% saline, 0.9% isotonic saline, 0.3% glycine, 5%dextrose, and the like, and may include glycoproteins for enhancedstability, such as albumin, lipoprotein, globulin, etc. In someembodiments, normal buffered saline (135-150 mM NaCl) is used. Thecompositions can contain pharmaceutically acceptable auxiliarysubstances to approximate physiological conditions, such as pH adjustingand buffering agents, tonicity adjusting agents, wetting agents, e.g.,sodium acetate, sodium lactate, sodium chloride, potassium chloride,calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.Injection solutions and suspensions can also be prepared from sterilepowders, granules, and tablets. In some embodiments, the composition isadministered by intravenous infusion, topically, intraperitoneally,intravesically, or intrathecally. The Klotho polypeptide formulation canbe provided in unit-dose or multi-dose sealed containers, such asampoules and vials.

The Klotho polypeptide composition, alone or in combination with othersuitable components, can be made into aerosol formulations (“nebulized”)to be administered via inhalation. Aerosol formulations can be placedinto pressurized acceptable propellants, such asdichlorodifluoromethane, propane, and nitrogen.

The pharmaceutical preparation can be packaged or prepared in unitdosage form. In such form, the preparation is subdivided into unit dosescontaining appropriate quantities of the active component, e.g.,according to the dose of Klotho polypeptide. The unit dosage form can bea packaged preparation, the package containing discrete quantities ofpreparation. The composition can, if desired, also contain othercompatible therapeutic agents. In some embodiments, the Klothopolypeptide composition can be formulated in a kit for administration.

In some embodiments, a pharmaceutical composition comprising a klothopolypeptide is administered orally. In some embodiments, apharmaceutical composition comprising a klotho polypeptide isadministered mucosally, e.g., nasally. In some embodiments, apharmaceutical composition comprising a klotho polypeptide isadministered by injection, e.g., subcutaneous, intraperitoneal,intravenous, or intramuscular. In some embodiments, a pharmaceuticalcomposition comprising a klotho polypeptide is administered by infusion,e.g., using a reservoir or osmotic minipump.

An example of administration of a pharmaceutical composition includesstoring the Klotho polypeptide at 10 mg/ml in sterile isotonic aqueoussaline solution at 4° C., and diluting it in an appropriate solution forinjection prior to administration to the patient. In some embodiments,the Klotho polypeptide composition can be administered by intravenousinfusion over the course of 0.25-2 hours. In some embodiments, theadministration procedure is via bolus injection.

In therapeutic use, the Klotho polypeptide can be administered at theinitial dosage of about 0.1 μg/kg to about 1000 μg/kg daily and adjustedover time. A daily dose range of about 1 μg/kg to about 500 μg/kg, orabout 10 μg/kg to about 100 μg/kg, or about 30 μg/kg to about 50 ug/kgcan be used. The dosage is varied depending upon the requirements of thepatient, the severity of the condition being treated, and the route ofadministration. For example, for injection of Klotho polypeptide, theeffective dose is typically in the range of 10-100 μg/kg, while fordirect delivery to the central nervous system (CNS), the effectivedosage is lower, e.g., 5-30 μg/kg. For oral administration, theeffective dose is higher, e.g., in the range of 50-10,000 μg/kg (e.g.,100 μg/kg-2 mg/kg). The dose is chosen in order to provide effectivetherapy for the patient. The dose may be repeated at an appropriatefrequency which may be in the range of once or twice per day, once ortwice per week to once every three months, depending on thepharmacokinetics of the Klotho polypeptide composition (e.g., half-lifein the circulation) and the pharmacodynamic response (e.g., the durationof the therapeutic effect).

Administration can be periodic. Depending on the route ofadministration, the dose can be administered, e.g., once every 1, 3, 5,7, 10, 14, 21, or 28 days or longer (e.g., once every 2, 3, 4, or 6months). In some cases, administration is more frequent, e.g., 2 or 3times per day. The patient can be monitored to adjust the dosage andfrequency of administration depending on therapeutic progress and anyadverse side effects, as will be recognized by one of skill in the art.

Dosages can be empirically determined considering the type and severityof cognitive condition diagnosed in a particular patient. The doseadministered to a patient, in the context of the present disclosure,should be sufficient to affect a beneficial therapeutic response in thepatient over time. The size of the dose will also be determined by theexistence, nature, and extent of any adverse side-effects that accompanythe administration of any particular composition in a particularpatient, as will be recognized by the skilled practitioner.

In some embodiments, the Klotho polypeptide composition is administeredto an (e.g., human) individual having at least normal cognitivefunction. As described herein, it has been surprisingly discovered thatnot only can Klotho improve cognition in individuals with impairedcognition, Klotho can also improve cognition of individuals with atleast normal cognition. Thus in some embodiments, the individualreceiving the Klotho polypeptide composition begins initially with atleast normal cognition and following administration of the Klothopolypeptide composition attains improved cognition compared to theinitial level of cognition. The level of cognition of an individual canbe determined as is known in the art. Normal cognitive functions aredetermined by scores from sets of cognitive tests that are compiled intoglobal cognitive scores, as described in Dubal D B et al. (2014) CellReports 7:1065-1076. Such cognition tests include tests of executivefunction and working memory such as Trails A and Trails B (Dubal D B etal. (2014) Cell Reports 7:1065-1076). In some embodiments,administration of Klotho results in an improvement of cognition (whetherinitially at least normal or impaired), by at least 5%, 10%, 20% ormore.

In some embodiments, administration results in improved motor function.In some embodiments, the Klotho polypeptide composition is administeredto an (e.g., human) individual having impaired motor function. Forexample, in some embodiments, the individual has stroke to the brain orspinal cord (ischemic or hemorrhagic), neurodegenerative disease(Parkinson's disease, Lewy body dementia, multiple system atrophy,amyotropic lateral sclerosis, prion disorder, Huntington's disease,supranuclear palsy), Parkinsonism, traumatic brain injury,neuroinfectious brain lesions, multiple sclerosis and related autoimmuneand demyelinating disease, spinal cord lesions (compressive, infectious,toxic or metabolic, autoimmune, oncologic), brain tumor, epilepsy,paraneoplastic disorder, neurodevelopmental disorder (mitochondrial,autosomal genetic), muscle disease (polymyositis, dermatomyositis,inclusion body myositis, infectious, endocrine, metabolic, toxic,congenital myopathy, congenital muscular dystrophy, hereditary),neuropathies (Guillain-Barre syndrome, axonal and demyelinating,diabetic, toxic, metabolic, infectious, critical illness, entrapment),tick paralysis, myasthenia gravis, and spinal muscular atrophy. Changesin motor function can be assayed as known in the art. Exemplary motorfunction assays include but are not limited to electromyogram and nerveconduction studies, direct or device-assisted clinical testing ofstrength, tone, and muscle bulk, reflex examination, coordinationexamination, and gait analysis. Assays for testing etiologies causingdeficits of motor function include but are not limited to magneticresonance imaging of the central nervous system, muscle biopsy, nervebiopsy, and laboratory studies.

Thus in some embodiments, additional administration is dependent onpatient progress, e.g., the patient is monitored betweenadministrations. For example, after the first administration or round ofadministrations, the patient can be monitored for cognitive ability orfor side effects, e.g., weakness, dizziness, nausea, etc.

In some embodiments, the individual has a chronic condition, so thatklotho is administered over an indefinite period, e.g., for the lifetimeof the patient. In such cases, administration is typically periodic.Diseases that are considered long-term or chronic include, but are notlimited to Alzheimer's disease, Parkinson's disease, Huntington'sdisease, and cognitive decline associated with hypertension and heartdisease.

In some embodiments, the Klotho polypeptide is linked to a stabilizingmoiety such as PEG, glycosylation, or a liposome or other nanocarrier.U.S. Pat. Nos. 4,732,863 and 7,892,554 and Chattopadhyay et al. (2010)Mol Pharm 7:2194 describe methods for attaching a polypeptide to PEG,PEG derivatives, and nanoparticles (e.g., liposomes). Liposomescontaining phosphatidyl-ethanolamine (PE) can be prepared by establishedprocedures as described herein. The inclusion of PE provides an activefunctional site on the liposomal surface for attachment. In someembodiments, the Klotho polypeptide is linked to an affinity tag, e.g.,a histidine tag (e.g., 4-16 histidine residues), streptavidin, or anantibody target.

The Klotho polypeptide can also be formulated as a sustained-releasepreparation (e.g., in a semi-permeable matrices of solid hydrophobicpolymers (e.g., polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly (vinylalcohol)), polylactides.The Klotho polypeptide can be entrapped in a nanoparticle prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nanoparticles and nanocapsules) or in macroemulsions.

In some embodiments, the Klotho polypeptide is labeled, e.g., fortracking in the body or ex vivo. The Klotho polypeptide can be labeledany diagnostic agent known in the art, as provided, for example, in thefollowing references: Armstrong et al., Diagnostic Imaging, 5^(th) Ed.,Blackwell Publishing (2004); Torchilin, V. P., Ed., Targeted Delivery ofImaging Agents, CRC Press (1995); Vallabhajosula, S., Molecular Imaging:Radiopharmaceuticals for PET and SPECT, Springer (2009). The diagnosticagent can be detected by a variety of ways, including as an agentproviding and/or enhancing a detectable signal. Detectable signalsinclude, but are not limited to, gamma-emitting, radioactive, echogenic,optical, fluorescent, absorptive, magnetic, or tomography signals.Techniques for imaging the diagnostic agent can include, but are notlimited to, single photon emission computed tomography (SPECT), magneticresonance imaging (MRI), optical imaging, positron emission tomography(PET), computed tomography (CT), x-ray imaging, gamma ray imaging, andthe like. The terms “detectable agent,” “detectable moiety,” “label,”“imaging agent,” and like terms are used synonymously herein.

In some embodiments, the label can include optical agents such asfluorescent agents, phosphorescent agents, chemiluminescent agents, andthe like. Numerous agents (e.g., dyes, probes, labels, or indicators)are known in the art and can be used in the present invention. (See,e.g., Invitrogen, The Handbook—A Guide to Fluorescent Probes andLabeling Technologies, Tenth Edition (2005)). Fluorescent agents caninclude a variety of organic and/or inorganic small molecules or avariety of fluorescent proteins and derivatives thereof. For example,fluorescent agents can include but are not limited to cyanines,phthalocyanines, porphyrins, indocyanines, rhodamines, phenoxazines,phenylxanthenes, phenothiazines, phenoselenazines, fluoresceins,benzoporphyrins, squaraines, dipyrrolo pyrimidones, tetracenes,quinolines, pyrazines, corrins, croconiums, acridones, phenanthridines,rhodamines, acridines, anthraquinones, chalcogenapyrylium analogues,chlorins, naphthalocyanines, methine dyes, indolenium dyes, azocompounds, azulenes, azaazulenes, triphenyl methane dyes, indoles,benzoindoles, indocarbocyanines, benzoindocarbocyanines, and BODIPY™derivatives. Fluorescent dyes are discussed, for example, in U.S. Pat.Nos. 4,452,720, 5,227,487, and 5,543,295.

The label can also be a radioisotope, e.g., radionuclides that emitgamma rays, positrons, beta and alpha particles, and X-rays. Suitableradionuclides include but are not limited to ²²⁵Ac, ⁷²As, ²¹¹At, ¹¹B,¹²⁸Ba, ²¹²Bi, ⁷⁵Br, ⁷⁷Br, ¹⁴C, ¹⁰⁹Cd, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ¹⁸F, ⁶⁷Ga, ⁶⁸Ga,³H, ¹⁶⁶Ho, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³⁰I, ¹³¹I, ¹⁷⁷Lu, ¹³N, ¹⁵O, ³²P, ³³P,²¹²Pb, ¹⁰³Pd, ¹⁸⁶Re, ¹⁸⁸Re, ⁴⁷Sc, ¹⁵³Sm, ⁸⁹Sr, ^(99m)Tc, ⁸⁸Y and ⁹⁰Y. Insome embodiments, radioactive agents can include ¹¹¹In-DTPA,^(99m)Tc(CO)₃-DTPA, ^(99m)Tc(CO)₃-ENPy2, ^(62/64/67)Cu-TETA,^(99m)Tc(CO)₃-IDA, and ^(99m)Tc(CO)₃ triamines (cyclic or linear). Insome embodiments, the agents can include DOTA and its various analogswith ¹¹¹In, ¹⁷⁷Lu, ¹⁵³Sm, ^(88/90) Y, ^(62/64/67)Cu, or ^(67/68)Ga. Insome embodiments, a nanoparticle can be labeled by incorporation oflipids attached to chelates, such as DTPA-lipid, as provided in thefollowing references: Phillips et al., Wiley Interdisciplinary Reviews:Nanomedicine and Nanobiotechnology, 1(1): 69-83 (2008); Torchilin, V. P.& Weissig, V., Eds. Liposomes 2nd Ed.: Oxford Univ. Press (2003);Elbayoumi, T. A. & Torchilin, V. P., Eur. J. Nucl. Med. Mol. Imaging33:1196-1205 (2006); Mougin-Degraef, M. et al., Int'l J. Pharmaceutics344:110-117 (2007).

In some embodiments, the diagnostic agent can be associated with asecondary binding ligand or to an enzyme (an enzyme tag) that willgenerate a colored product upon contact with a chromogenic substrate.Examples of suitable enzymes include urease, alkaline phosphatase,(horseradish) hydrogen peroxidase and glucose oxidase. Secondary bindingligands include, e.g., biotin and avidin or streptavidin compounds asknown in the art.

V. Cognitive Conditions and Disorders

Klotho polypeptides (and functional variants and fragments thereof) canbe administered to improve cognition for a number of conditions andsituations. This includes treatment of individuals with lower thannormal or declining cognitive ability, or prophylactic treatment ofindividuals in need of improved or increased cognitive ability.

Klotho polypeptides (and functional variants and fragments thereof) canbe used to prevent or reduce cognitive decline associated with aging,e.g. in individuals 50 years of age or older, or upon initial signs ofcognitive decline.

Klotho polypeptides (and functional variants and fragments thereof) canalso be used to treat individuals with age-related, non-age related, ordisease related conditions including, but not limited to:

Neurodegenerative diseases and dementia: Alzheimer's disease,Parkinson's disease, Huntington's disease, frontotemporal dementia,progressive supranuclear palsy, corticobasalar degeneration, mildcognitive impairment, vascular dementia, Lewy body dementia, amyotropiclateral sclerosis, prion disorder, HIV-related dementia;

Mental or mood disorders: depression, schizophrenia, attentiondeficit/hyperactivity disorder, autism spectrum disorder, intellectualdisability, a mood disorder, and a psychotic disorder;

Childhood neurodevelopmental syndromes and brain tumors: X-linked mentaldisability or retardation, astrocytoma, ependymoma, medulloblastoma,oligodendroglioma;

Genetic syndromes affecting learning: Down's syndrome, Angelman'ssyndrome, Rett's syndrome;

Metabolic disorders affecting cognition: phenylketonuria, Lesch-Nyhan,galactosemia, and adrenoleukodystrophy;

Cognitive decline associated with chemotherapy and/or radiation therapy;and

Additional conditions and disorders: pain-associated cognitive effects,traumatic brain injury, stroke, multiple sclerosis, neuroautoimmunedisease, epilepsy, delirium, paraneoplastic disorder, developmentaldelay, and leukodystrophies.

Klotho polypeptides (and functional variants and fragments thereof) canbe also be administered to provide increased cognition for individualsdesiring improved cognition, e.g., individuals exposed to stress, sleepdeprivation, or jet lag, or for individuals requiring superior cognitivefunction, such as surgeons, air-traffic controllers, and militarypersonal. In such cases, the klotho polypeptide composition can beadministered 2-24 hours before the desired effect, which can last about3-5 days for working memory and about 2 weeks for spatial memory.

Cognitive ability can be measured using any method known in the art,e.g., for testing memory, language ability, executive functions,visuospatial function, dementia, or multi-parameter neuropsychologicalabilities. In some embodiments, Klotho administration results in atleast a 1%, 2%, 5%, 7%, 10%, 15%, 20%, 30%, 50%, or greater improvementin score on a standard cognitive ability test (e.g., measured 1-3 daysafter administration). In some embodiments, the testing is carried outmore than once for an individual, e.g., one or more time over the courseof treatment with Klotho.

For example, standard tests for memory and learning can be applied,e.g., to determine semantic, episodic, procedural, priming, and/orworking (i.e., short term) memory. Common tests include Cambridgeprospective memory test (CAMPROMPT), memory assessment scales (MAS), Reyauditory verbal learning test, Rivermead behavioral memory test, Test ofmemory and learning (TOMAL), Wechsler memory scale (WMS), and Test ofmemory malingering (TOMM). Tests for language functions include, e.g.,Boston Diagnostic Aphasia Examination (BDAE), Comprehensive aphasia test(CAT), and Multilingual aphasia examination (MAE).

Executive function (e.g., problem solving, planning, organization,inhibitory control) can be tested using Behavioral assessment ofdysexecutive syndrome (BADS), CNS vital signs (Brief Core Battery),Controlled oral word association test (COWAT), Delis-Kaplan ExecutiveFunction System (D-KEFS), Digit vigilance test, Kaplan Baycrestneurocognitive assessment (KBNA), Hayling and Brixton tests, Tests ofvariables of attention (TOVA), Wisconsin card sorting test (WCST), orTest of everyday attention (TEA). Visuospatial ability (e.g., visualperception, construction and integration) can be tested using the ClockTest, Hooper visual organization task (VOT), or Rey-Osterrieth complexfigure tests. Dementia can be quantified using the clinical dementiarating or dementia rating scale.

Multi-parameter tests for neuropsychological function (e.g., cognitivefunction) include but are not limited to the Barcelonaneuropsychological test (BNT), Cambridge neuropsychological testautomated battery (CANTAB), Cognistat, Cognitive assessment screeninginstrument (CASI), Cognitive function scanner (CFS), Dean-Woodcockneuropsychology assessment system (DWNAS), General practitionalassessment of cognition (GPCOG) Mini mental state examination (MMSE),NEPSY, or the CDR computerized assessment system.

Alternatively, cognition can be determined using structural or molecularproxies for cognitive activity, e.g., compared over time to detectchanges. Cognitive changes can be detected, e.g., by observing changesto brain structure, connectivity, activation, inhibition, or synapticplasticity, e.g., by MRI, fMRI, EEG, TMS and TES, and/or any combinationof these. In some embodiments, brain activity is observed. In someembodiments, Klotho administration results in a 1.5-fold, 2-fold,5-fold, 7-fold, 10-fold, or greater increase in brain activity (e.g.,measured 1-3 days after administration). Molecular proxies for improvedcognition include, but are not limited to: increased levels of GluN2B,increased GluN2B synaptic localization, increased NMDA receptoractivation, and/or increased c-fos activation in the brain. Thesemeasures are particularly relevant to cognition. Such method caninclude, e.g., obtaining a sample of neuronal tissue or CSF from anindividual and using standard assays to determine gene expression oractivation.

Similarly, in mice and other non-human animals, cognitive ability can betested with measures of executive function (working memory, attention,processing speed, set shifting), visiospatial learning and memory,object memory, pattern recognition, fear memory, passive avoidancememory, habituation, and novel object recognition, for example. Commontests include but are not limited to the Morris water maze, Barnes maze,radial arm water maze, y-maze, T-maze, and open field habituation. Brainimaging techniques are similarly applicable.

VI. EXAMPLES Example 1

A. Materials and Methods

Mice. All mice were on a C57BL/6J background and were kept on a 12-hlight/dark cycle with ad libitum access to food (Picolab Rodent Diet 20,Labdiet) and water. The standard housing group was five mice per cageexcept for single housing during water maze studies. All cognitive andbehavioral studies were carried out during the light cycle. All studieswere conducted in a blinded manner.

Treatments. Vehicle or klotho (recombinant mouse klotho, amino acids35-982, with a C-terminal His tag, R&D Systems) was injectedintraperitoneally (i.p.) before behavioral testing of mice.

Morris water maze cognitive behavior test. Water maze studies werecarried out as described in Dubal et al. (2014) Cell Reports 7:1065;Dubal et al. (2015) Journal of Neuroscience 35:2358. Mice were treatedwith either vehicle or klotho (10 μg/kg) i.p. 4 h prior to testing dailyfor 5 days. Briefly, mice were tested in a pool (diameter, 122 cm) withwhite, opaque water (21°±1° C.). A square, 14-cm2 platform was 2 cmbelow the surface. Before hidden platform training, mice underwent twopre-training trials by swimming through a channel to mount a hiddenplatform. Over the course of hidden platform training, the platformlocation stayed consistent while the drop location was varied betweentrials. Mice underwent two training sessions, consisting of two trialsof 60 s each, daily for four days. For the probe trial testing, theplatform was removed and the mice were allowed to swim for 60 s. After 1h and 24 h probe trials, mice were tested for their ability to find avisible platform marked with a cue (15-cm pole on the platform) over twosessions. As part of the studies, swim velocities were also recorded.

Y-maze cognitive behavior test. Y-maze studies were carried out asdescribed in Dubal et al. (2014) Cell Reports 7:1065. Mice were treatedwith either vehicle or klotho (10 μg/kg) i.p. 18 h prior to testing.Briefly, mice were acclimated to the room 30 min prior to testing. Then,mice were placed in one arm of the Y-maze (three identical arms, 120°apart) and explored for 4 min. Arm entries were recorded and analternation was noted any time the mouse entered each of the three armsin successive arm entries; chance alternation was 22%. The apparatus wascleaned with 70% alcohol between testing sessions. Percent alternationwas calculated from recorded data.

B. Systemic Klotho Delivery Enhances Working Memory in Young Mice

To investigate whether therapeutic delivery of klotho can enhancecognition, we injected mice with recombinant klotho (i.p.) and 18 hlater, tested working memory in the Y-maze. Compared to vehicle-treatedmice, klotho-treated mice showed more alternations, indicating superiorworking memory (FIG. 1A). Thus, systemic klotho delivery enhancedworking memory, a process that involves frontal cortical brain regions.

C. Systemic Klotho Delivery Enhances Spatial Learning in Young Mice

We next tested whether klotho treatment enhances spatial learning in theMorris water maze. We injected mice with recombinant klotho (i.p.) 4 hprior to testing daily for five days in a group of mice (Cohort 1).Compared to vehicle-treated mice, klotho-treated mice performed betterin spatial learning of the hidden platform location (FIG. 1B). Bothgroups swam at equal speeds (FIG. 1C). In an independent group of mice(Cohort 2), we injected recombinant klotho (i.p.) 18 h prior to testingdaily. Again, compared to vehicle-treated mice, klotho-treated miceperformed better in spatial learning (FIG. 1D). Thus, systemic klothodelivery enhanced spatial learning, a process that involves frontalcortical and hippocampal brain regions.

D. Systemic Klotho Delivery Enhances Spatial Memory in Young Mice

To determine whether klotho treatment can enhance spatial memory, weremoved the platform and performed probe testing following hiddentraining in the Morris water maze. In probe trials of Cohort 1, spatialmemory retention was assessed by measuring the affinity of mice for thetarget area. In probe testing at 1 h, klotho-treated mice spent moretime at the target center (FIG. 1E), showed increased frequency ofcrossing the target (FIG. 1F), and increased time in the target quadrant(FIG. 1G), compared to vehicle-treated controls. In probe testing at 24h, klotho-treated mice continued to show more center duration (FIG. 1H),crossing frequency (FIG. 10 , and percent time in the target quadrant(FIG. 1J), compared to vehicle-treated mice. Thus, systemic klothodelivery enhanced spatial memory, a process that engages hippocampalbrain regions.

The data show that systemic delivery of klotho enhances normal cognitionin mice. Klotho treatment improved learning and memory in multiple testsand measures including working memory, spatial learning, and spatialmemory. These findings provide a direct therapeutic application forboosting cognitive functions, a therapy that is relevant, but notlimited to, cognitive enhancement of the normal brain and cognitivedysfunction due to normal aging, numerous neurodegenerative, neurologic,and psychiatric diseases, childhood developmental syndromes, traumaticbrain injury, and stress.

Example 2

Methods.

Mice. All mice were on a C57BL/6J background and were kept on a 12-hlight/dark cycle with ad libitum access to food (Picolab Rodent Diet 20,Labdiet) and water. The standard housing group was five mice per cageexcept for single housing during water maze studies. All motor,cognitive and behavioral studies were carried out during the lightcycle. All studies were conducted in a blinded manner.

Aged mice (18 months) were obtained from the NIH aging colony and wereused in experiments. Transgenic mice that model human neurodegenerativediseases related to α-synuclein toxicity were utilized in experiments;these mice express full length human α-synuclein from the mouse Thy-1promoter (Rockenstein E, et al. (2002) J Neurosci Res 68:568-578) andexhibit motor, cognitive and behavioral deficits. Increased expressionof the human α-synuclein protein contributes to severalneurodegenerative diseases in the human condition including, but notlimited to, Parkinson's disease (PD), Alzheimer's disease (AD), Lewybody dementia (LBD), and multiple system atrophy (MSA).

Treatments. Vehicle or α-klotho (recombinant mouse α-klotho, amino acids35-982, with a C-terminal His tag, R&D Systems) was injectedintraperitoneally (i.p.) before behavioral testing of mice as indicated.All animal studies were approved by the Institutional Animal Care andUse Committee of the University of California, San Francisco andconducted in compliance with NIH guidelines.

Cognitive and Motor Behavior

Morris water maze. Water maze studies were carried out as described(Zarei M, et al. (2013) J Neurol Neurosurg Psychiatry 84:875-881; DubalD B et al. (2014) Cell Reports 7:1065-1076). Mice were treated witheither vehicle or klotho (10 μg/kg) i.p. 4 h prior to testing daily for5 days. Briefly, mice were tested in a pool (diameter, 122 cm) withwhite, opaque water (21°±1° C.). A square, 14-cm2 platform was 2 cmbelow the surface. Before hidden platform training, mice underwent twopre-training trials by swimming through a channel to mount a hiddenplatform. Over the course of hidden platform training, the platformlocation stayed consistent while the drop location was varied betweentrials. Mice underwent two training sessions, consisting of two trialsof 60 s each, daily for four days. For the probe trial testing, theplatform was removed and the mice were allowed to swim for 60 s. After 1h and 24 h probe trials, mice were tested for their ability to find avisible platform marked with a cue (15-cm pole on the platform) over twosessions. As part of the studies, swim velocities were also recorded.

Y-maze. Y-maze studies were carried out as described (Dubai D B et al.(2014) Cell Reports 7:1065-1076). Mice were treated with either vehicleor klotho (10n/kg) i.p. 18 h prior to testing. Briefly, mice wereacclimated to the room 30 min prior to testing. Then, mice were placedin one arm of the Y-maze (three identical arms, 120° apart) and exploredfor 4 min. Arm entries were recorded and an alternation was noted anytime the mouse entered each of the three arms in successive arm entries;chance alternation was 22%. The apparatus was cleaned with 70% alcoholbetween testing sessions. Percent alternations was calculated fromrecorded data.

Large Y-maze. Large Y-maze studies were performed as described (Dellu F,et al. (1992) Brain Res 588:132-139) with minor modifications. The largeY-maze apparatus consists of three identical arms, 120° apart, with adistinct and different visual cue at the back end of two arms; the thirdarm without a visual cue is the start arm. Prior to training or testing,mice were acclimated to the dimly lit room for 60 minutes. Duringtraining, one arm with a visual cue was blocked with a solid divider andmice were placed at the end of the start arm. Mice were allowed toexplore the open arm for five minutes and then returned to their cages.The open arm used in training (the familiar arm) was counterbalancedthroughout the cohort testing. 16 hours after training, mice underwenttesting. During testing, the divider was removed. Mice were placed inthe start arm and allowed to explore the arms with novel and familiarvisual cues (novel arm and familiar arm) for 5 minutes. Number ofentries and duration of time spent in the novel and familiar arms wasrecorded and measured. The ratio of novel to familiar arm entries orduration was calculated to assess spatial and working memory. Theapparatus was cleaned with 70% alcohol between sessions.

Rota Rod. Mice were acclimated to the room 60 minutes prior to eachsession. Five mice were simultaneously put on the rotating rod (RotaRod, Med Associates Inc, VT) at a constant speed of 16 rpm for a maximumof 300 seconds. Latency to fall was recorded in each trial. Three trialswere performed consecutively with a 10 minute rest between trials. Theapparatus was cleaned with 70% alcohol between testing sessions.

Open field. Total activity in the open field was measured as described(Dubal D B, et al. (2015) J Neurosci 35:2358-2371) with an automatedFlex-Field/Open Field Photobeam Activity System (San Diego Instruments,San Diego, Calif.). Mice were acclimated to the testing room for 30minute and then tested in a clear plastic chamber (41×30 cm) for 5 min,with two photobeam arrays measuring movements. The apparatus was cleanedwith 70% alcohol between testing sessions.

Results.

Systemic klotho delivery enhances cognition in aged mice. To determinewhether therapeutic delivery of klotho can enhance cognition in agedmice, we injected mice with recombinant klotho (i.p., 10 μg/kg) once andthen trained them for a task to measure working and spatial memory inthe large Y-maze 24 h later. Then, 18 h following training (42 h afterklotho delivery), mice underwent testing. At baseline, and withoutklotho treatment, the aged mice did not show novel arm preference,indicating a cognitive deficit. Compared to vehicle-treated mice,klotho-treated mice showed a persistent preference for the novel armlargely throughout exploration (FIG. 2A,B), indicating better cognitionthat involves frontal and hippocampal brain regions. These data showthat klotho enhances cognition in mice with age-induced cognitivedeficits. These data suggest that therapeutic delivery of klothoenhances cognition in the aging brain.

Systemic klotho delivery enhances cognition in a long-lasting manner. Todetermine whether therapeutic delivery of klotho can induce long-lastingcognitive enhancement, in a manner that extends beyond its half-life of7 hours (Hu M C, et al. (2015) J Am Soc Nephrol.), we tested young miceapproximately two weeks after 5 days of daily klotho treatment andcognitive testing (i.p., 0.5 or 2.5 μg/kg). As expected, vehicle-treatedmice did not show preference for the novel arm, indicating a loss ofmemory for the visual cue after two weeks. In contrast, klotho-treatedmice showed a clear preference for the novel arm of the maze—even twoweeks after the last treatment and in a dose-dependent manner (FIG. 3 ).These data indicate that klotho-mediated cognitive enhancement innormal, young mice was long-lasting and extended at least two weeksbeyond its half-life. These data suggest that therapeutic delivery ofklotho induces organizational changes in the brain that enhance neuralfunction.

Systemic klotho delivery improves cognitive dysfunction in mice thatmodel neurodegenerative disease. We next tested whether acute,therapeutic delivery of klotho can improve existing cognitive deficitsin a mouse model of neurodegenerative disease. In thiswell-characterized model of disease, mice express human α-synuclein(hSYN), a pathogenic protein that causes cognitive and motor deficitsand contributes to Parkinson's disease, Alzheimer's disease, Lewy bodydementia, and multiple system atrophy. In these mice, cognitive functionwas assessed in the large-Y maze. This task is optimal since it utilizesthe mouse's natural tendency to explore, avoids testing-inducedstressors, and is not affected by motor difficulties that limit othertests (such as inability to swim appropriately in a water maze).Compared to nontransgenic (NTG) mice, hSYN mice showed decreasedpreference for the novel arm, as shown by decreased duration (FIG. 4A).Remarkably, klotho treatment of hSYN mice (i.p., 2.5 μg/kg) increasednovel arm preference (FIG. 4A,B), indicating acute and therapeuticcognitive improvement. There was an overall effect of klotho treatmentin enhancing memory as indicated by increasing novel arm preference(Two-way ANOVA KL effect p<0.05) (FIG. 4B). These data suggest thattherapeutic delivery of klotho can reverse or improve cognitive deficitscaused by α-synuclein toxicity in neurodegenerative diseases. Of note,klotho-mediated cognitive enhancement could contribute to improved motorlearning.

Systemic klotho delivery improves early motor dysfunction in mice thatmodel neurodegenerative disease. Since α-synculein toxicity inducesdeficits in motor function, a major clinical problem inneurodegenerative diseases, we tested whether therapeutic delivery ofklotho can improve this key measure in hSYN mice. Motor function wasassessed on a spinning rod, the Rota rod. Compared to NTG mice, hSYNmice showed decreased motor function (FIG. 5 ). Remarkably, klothotreatment (i.p., 2.5 μg/kg) of hSYN mice acutely increased motorfunction, as shown by longer latency on the spinning rod (FIG. 5 ).These data indicate that klotho treatment acutely improves early motorfunction in hSYN mice. These data suggest that therapeutic delivery ofklotho can improve deficits related to motor problems caused byα-synuclein toxicity in neurodegenerative diseases.

Systemic klotho delivery does not alter total movements or activity ofmice. To examine whether klotho delivery alters total movements andactivity of mice, we examined mouse exploration of an open field. Totalmovements did not differ between Veh- or KL-treated (i.p., 2.5 μg/kg)mice (FIG. 6 ). These data suggest that the therapeutic effects ofklotho are specific to cognitive and motor functions and are notinfluenced by non-specific actions such as hyperactivity.

Discussion

Our data show that systemic delivery of klotho, which does not cross theblood brain barrier, enhances normal cognition in young mice in a mannerthat is long-lasting, improves cognitive deficits in normal young miceand in aging mice; it also improves cognitive and motor deficits intransgenic mice that model major neurodegenerative diseases such asAlzheimer's disease, Parkinson's disease, Lewy body dementia, andmultiple system atrophy.

These findings provide a direct therapeutic application for boostingcognitive functions in the normal brain and improving brain function inaging and neurodegenerative disease. This therapeutic application isalso relevant, but not limited to, cognitive dysfunction due to numerousneurologic, and psychiatric diseases, childhood developmental syndromes,traumatic brain injury, and stress.

Our data show that:

-   1. Systemic administration of mouse recombinant klotho forms enhance    normal cognition across the lifespan from young (2-7 mos) to aged    (18 mos) male and female mice.-   2. Systemic klotho therapy is effective in enhancing cognition when    given from 4 hours to 16 days prior to testing. The    cognitive-enhancing effects last for at least two weeks following    extensive cognitive training.-   3. Systemic doses of mouse klotho between 0.5 μg/kg and 10 μg/kg    enhance cognition in conditions tested.-   4. Systemic therapy with recombinant klotho enhances cognition in a    mouse model of neurodegenerative disease relevant to, but not    limited to, Alzheimer's disease, Parkinson's disease, Lewy body    dementia, and multiple system atrophy.-   5. In addition to improving cognitive deficits in a mouse model of    neurodegenerative disease, klotho therapy also improved early motor    function.-   6. The therapeutic effects of klotho on enhancing cognition extend    far beyond its half-life of approximately 7 hours.-   7. The therapeutic effects of klotho appear specific to cognitive    and motor functions and are not influenced by non-specific actions    such as hyperactivity.

The above examples are provided to illustrate the invention but not tolimit its scope. Other variants of the invention will be readilyapparent to one of ordinary skill in the art and are encompassed by theappended claims. All publications, databases, internet sources, patents,patent applications, and accession numbers cited herein are herebyincorporated by reference in their entireties for all purposes.

VII. Informal Sequence Listing

SEQ ID NO: 1: Human Klotho Protein      10         20         30         40MPASAPPRRP RPPPPSLSLL LVLLGLGGRR LRAEPGDGAQ      50           60         70         80TWARFSRPPA PEAAGLFQGT FPDGFLWAVG SAAYQTEGGW        90        100        110        120QQHGKGASIW DTFTHHPLAP PGDSRNASLP LGAPSPLQPA       130        140        150        160TGDVASDSYN NVFRDTEALR ELGVTHYRFS ISWARVLPNG       170        180        190        200SAGVPNREGL RYYRRLLERL RELGVQPVVT LYHWDLPQRL       210        220        230        240QDAYGGWANR ALADHFRDYA ELCFRHFGGQ VKYWITIDNP       250        260        270        280YVVAWHGYAT GRLAPGIRGS PRLGYLVAHN LLLAHAKVWH       290        300        310        320LYNTSFRPTQ GGQVSIALSS HWINPRRMTD HSIKECQKSL       330        340        350        360DFVLGWFAKP VFIDGDYPES MKNNLSSILP DFTESEKKFI       370        380        390        400KGTADFFALC FGPTLSFQLL DPHMKFRQLE SPNLRQLLSW       410        420        430        440IDLEFNHPQI FIVENGWFVS GTTKRDDAKY MYYLKKFIME       450        460        470        480TLKAIKLDGV DVIGYTAWSL MDGFEWHRGY SIRRGLFYVD       490        500        510        520FLSQDKMLLP KSSALFYQKL IEKNGFPPLP ENQPLEGTFP       530        540        550        560CDFAWGVVDN YIQVDTTLSQ FTDLNVYLWD VHHSKRLIKV       570        580        590        600DGVVTKKRKS YCVDFAAIQP QIALLQEMHV THFRFSLDWA       610        620        630        640LILPLGNQSQ VNHTILQYYR CMASELVRVN ITPVVALWQP       650        660        670        680MAPNQGLPRL LARQGAWENP YTALAFAEYA RLCFQELGHH       690        700        710        720VKLWITMNEP YTRNMTYSAG HNLLKAHALA WHVYNEKFRH       730        740        750        760AQNGKISIAL QADWIEPACP FSQKDKEVAE RVLEFDIGWL       770        780        790        800AEPIFGSGDY PWVMRDWLNQ RNNFLLPYFT EDEKKLIQGT       810        820        830        840FDFLALSHYT TILVDSEKED PIKYNDYLEV QEMTDITWLN       850        860        870        880SPSQVAVVPW GLRKVLNWLK FKYGDLPMYI ISNGIDDGLH       890        900        910        920AEDDQLRVYY MQNYINEALK AHILDGINLC GYFAYSFNDR       930        940        950        960TAPRFGLYRY AADQFEPKAS MKHYRKIIDS NGFPGPETLE       970        980        990       1000RFCPEEFTVC TECSFFHTRK SLLAFIAFLF FASIISLSLI       1010 FYYSKKGRRS YK

What is claimed is:
 1. A method of treating cognitive decline in anindividual having a neurodegenerative disease or traumatic brain injury(TBI), comprising administering systemically to the individual aneffective amount of a recombinant polypeptide comprising the amino acidsequence having at least 95% identity to the sequence of amino acids34-979 of SEQ ID NO:1, with the proviso that the polypeptide is not aFGF fusion protein.
 2. The method of claim 1, wherein the individual'sneurodegenerative disease is Alzheimer's disease, Parkinson's disease,frontotemporal dementia, mild cognitive impairment, or Lewy bodydementia.
 3. The method of claim 1, wherein the amino acid sequence isat least 99% identical to amino acids 34-979 of SEQ ID NO:1.
 4. Themethod of claim 1, wherein the amino acid sequence comprises amino acids34-979 of SEQ ID NO:1.
 5. The method of claim 1, comprising: determiningcognitive function in the subject before administration of thepolypeptide, determining cognitive function in the subject followingsystemic administration of the polypeptide, and comparing the cognitivefunction in the subject before administration of the klotho to thecognitive function of the subject following administration of thepolypeptide.
 6. The method of claim 1, wherein the polypeptide isadministered to the subject intravenously.
 7. The method of claim 2,wherein the individual's neurodegenerative disease is Alzheimer'sDisease.
 8. The method of claim 2, wherein the individual'sneurodegenerative disease is Parkinson's Disease.
 9. The method of claim2, wherein the individual's neurodegenerative disease is frontotemporaldementia.
 10. The method of claim 2, wherein the individual'sneurodegenerative disease is mild cognitive impairment.
 11. The methodof claim 2, wherein the individual's neurodegenerative disease is Lewybody dementia.
 12. The method of claim 1, wherein the individual hastraumatic brain injury.